Abstract
The effects of hypoxia and perfluorooctane sulfonamide (PFOSA), given singly and also in combination on endocrine, biotransformation, and oxidative stress responses were investigated in primary culture of salmon hepatocytes. Hypoxia was induced chemically using cobalt chloride (CoCl2) or deferroxamine (DFO). Primary culture of salmon hepatocytes were exposed to either CoCl2 (150 μM) or DFO (100 μM), in the presence or absence of PFOSA at 0, 25, and 50 μM for 24 and 48 h. Changes in transcript levels were analyzed by quantitative (real-time) PCR using gene-specific primers. CYP, catalase, GST, and SOD activities were analyzed spectrophotometrically. The hif-1α mRNA was used to validate cellular hypoxic condition, showing significantly induced transcription after 48-h exposure to DFO and CoCl2. Our data show that transcript levels for endocrine (ERα, Vtg, and Zrp), biotransformation (cyp1a, cyp3a, gst, and udpgt), and oxidative stress responses (catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr)) were differentially modulated by PFOSA and hypoxia alone, and these effects were dependent on the response parameters and time of exposure. In combined exposure scenarios, the observed effects were apparently hypoxia-dependent. However, the observed effects at transcript levels were not concomitant with those at functional protein levels, further emphasizing the potential differences that may exist between these biological levels. Biplot of principal component analysis (PCA) showed grouping of response variables after 48 h of exposure. The distribution of observations and variables indicate that PFOSA had little effect on most response variables, while clustering show a unique association between a given hypoxia condition (i.e., CoCl2 or DFO) in combination with PFOSA and transcripts, proteins, or enzyme activities.
Similar content being viewed by others
References
Anzenbacher P, Anzenbacherová E (2001) Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 58:737–747
Arukwe A (2006) Toxicological housekeeping genes: do they really keep the house? Environ Sci Technol 40:7944–9
Arukwe A, Goksoyr A (2003) Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp Hepatol 2:4
Arukwe A, Mortensen AS (2011) Lipid peroxidation and oxidative stress responses of salmon fed a diet containing perfluorooctane sulfonic- or perfluorooctane carboxylic acids. Comput Biochem Physiol C Toxicol Pharmacol 154:288–95
Arukwe A, Knudsen FR, Goksoyr A (1997) Fish zona radiata (eggshell) protein: a sensitive biomarker for environmental estrogens. Environ Health Perspect 105:418–22
Benninghoff AD, Bisson WH, Koch DC, Ehresman DJ, Kolluri SK, Williams DE (2011) Estrogen-like activity of perfluoroalkyl acids in vivo and interaction with human and rainbow trout estrogen receptors in vitro. Toxicol Sci 120:42–58
Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 91:179–94
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–54
Bradshaw TD, Trapani V, Vasselin DA, Westwell AD (2002) The aryl hydrocarbon receptor in anticancer drug discovery: friend or foe? Curr Pharm Design 8:2475–90
Bugel SM, White LA, Cooper KR (2010) Impaired reproductive health of killifish (Fundulus heteroclitus) inhabiting Newark Bay, NJ, a chronically contaminated estuary. Aquat Toxicol 96:182–93
Cech J, Crocker C (2002) Physiology of sturgeon: effects of hypoxia and hypercapnia. J Appl Ichthyol 18:320–324
Cheek AO, Landry CA, Steele SL, Manning S (2009) Diel hypoxia in marsh creeks impairs the reproductive capacity of estuarine fish populations. Mar Ecol Prog Ser 392:211–221
Cheng Y, Cui Y, Dang ZC, Xie WP, Li HS, Yin HH, Chen HM (2012) Effects of perfluorooctane sulfonate (PFOS) exposure on vitellogenin mRNA level in zebrafish (Brachydanio rerio). Huan Jing Ke Xue 33:1865–70
Chengelis CP (1988) Paradoxical effect of cobaltous chloride on carbon disulfide induced hepatotoxicity in rats. Res Commun Chem Pathol Pharmacol 61:83–96
Clarke DJ, Burchell B, George SG (1992) Differential expression and induction of UDP-glucuronosyltransferase isoforms in hepatic and extrahepatic tissues of a fish, Pleuronectes platessa: immunochemical and functional characterization. Toxicol Appl Pharmacol 115:130–6
Clearwater S, Pankhurst N (1997) The response to capture and confinement stress of plasma cortisol, plasma sex steroids and vitellogenic oocytes in the marine teleost, red gurnard. J Fish Biol 50:429–441
Clotfelter ED, Lapidus SJ, Brown AC (2013) The effects of temperature and dissolved oxygen on antioxidant defences and oxidative damage in the fathead minnow (Pimephales promelas). J Fish Biol 82:1086–92
Cochran RE, Burnett LE (1996) Respiratory responses of the salt marsh animals, Fundulus heteroclitus, Leiostomus xanthurus, and Palaemonetes pugio to environmental hypoxia and hypercapnia and to the organophosphate pesticide, azinphosmethyl. J Exp Mar Biol Ecol 195:125–144
Cooper RU, Clough LM, Farwell MA, West TL (2002) Hypoxia-induced metabolic and antioxidant enzymatic activities in the estuarine fish Leiostomus xanthurus. J Exp Mar Biol Ecol 279:1–20
Cui L, Liao CY, Zhou QF, Xia TM, Yun ZJ, Jiang GB (2010) Excretion of PFOA and PFOS in male rats during a subchronic exposure. Arch Environ Con Tox 58:205–213
Dery MAC, Michaud MD, Richard DE (2005) Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators. Int J Biochem Cell B 37:535–540
du Souich P, Fradette C (2011) The effect and clinical consequences of hypoxia on cytochrome P450, membrane carrier proteins activity and expression. Expert Opin Drug Metab Toxicol 7:1083–100
Du G, Hu J, Huang H, Qin Y, Han X, Wu D, Song L, Xia Y, Wang X (2013) Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine-related genes in vitro and in vivo. Environ Toxicol Chem 32:353–60
Fan J, Cai H, Yang S, Yan L, Tan W (2008) Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34+ cells. Comput Biochem Phys B 151:153–158
Fang C, Wu X, Huang Q, Liao Y, Liu L, Qiu L, Shen H, Dong S (2012) PFOS elicits transcriptional responses of the ER, AHR and PPAR pathways in Oryzias melastigma in a stage-specific manner. Aquat Toxicol 106–107, 9–19
Fivelstad S, Olsen AB, Kløften H, Ski H, Stefansson S (1999) Effects of carbon dioxide on Atlantic salmon (Salmo salar L.) smolts at constant pH in bicarbonate rich freshwater. Aquaculture 178:171–187
Fleming CR, Billiard SM, di Giulio RT (2009) Hypoxia inhibits induction of aryl hydrocarbon receptor activity in topminnow hepatocarcinoma cells in an ARNT-dependent manner. Comput Biochem Phys C 150:383–9
Fradette C, du Souich P (2003) Hypoxia-inducible factor-1 and activator protein-1 modulate the upregulation of CYP3A6 induced by hypoxia. Br J Pharmacol 140:1146–54
Fradette C, du Souich P (2004) Effect of hypoxia on cytochrome P450 activity and expression. Curr Drug Metab 5:257–71
Fradette C, Batonga J, Teng S, Piquette-Miller M, du Souich P (2007) Animal models of acute moderate hypoxia are associated with a down-regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and up-regulation of CYP3A6 and P-glycoprotein in liver. Drug Metab Dispos 35:765–71
George SG, Taylor B (2002) Molecular evidence for multiple UDP-glucuronosyltransferase gene families in fish. Mar Environ Res 54:253–257
Giesy JP, Kannan K (2001) Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol 35:1339–42
Giuliani ME, Benedetti M, Arukwe A, Regoli F (2013) Transcriptional and catalytic responses of antioxidant and biotransformation pathways in mussels, Mytilus galloprovincialis, exposed to chemical mixtures. Aquat Toxicol 134–135, 120–7
Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583–596
Gu YZ, Hogenesch JB, Bradfield CA (2000) The PAS superfamily: sensors of environmental and developmental signals. Annu Rev Pharmacol Toxicol 40:519–61
Guo M, Song LP, Jiang Y, Liu W, Yu Y, Chen GQ (2006) Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms. Apoptosis 11:67–77
Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139
Haddy J, Pankhurst N (1999) Stress-induced changes in concentrations of plasma sex steroids in black bream. J Fish Biol 55:1304–1316
Hagenaars A, Knapen D, Meyer IJ, van der Ven K, Hoff P, De Coen W (2008) Toxicity evaluation of perfluorooctane sulfonate (PFOS) in the liver of common carp (Cyprinus carpio). Aquat Toxicol 88:155–63
Hayes JD, McLellan LI (1999) Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 31:273–300
Houde M, De Silva AO, Muir DC, Letcher RJ (2011) Monitoring of perfluorinated compounds in aquatic biota: an updated review. Environ Sci Technol 45:7962–73
Ishimatsu A, Hayashi M, Lee KS, Kikkawa T, Kita J (2005) Physiological effects on fishes in a high-CO2 world. J Geophys Res. doi:10.1029/2004JC002564
Jo A, Ji K, Choi K (2014) Endocrine disruption effects of long-term exposure to perfluorodecanoic acid (PFDA) and perfluorotridecanoic acid (PFTrDA) in zebrafish (Danio rerio) and related mechanisms. Chemosphere 108:360–6
Kang JH, Asai D, Katayama Y (2007) Bisphenol A in the aquatic environment and its endocrine-disruptive effects on aquatic organisms. Crit Rev Toxicol 37:607–25
Kannan K, Corsolini S, Falandysz J, Fillmann G, Kumar KS, Loganathan BG, Mohd MA, Olivero J, Van Wouwe N, Yang JH, Aldoust KM (2004) Perfluorooctanesulfonate and related fluorochemicals in human blood from several countries. Environ Sci Technol 38:4489–95
Khan S, Liu SX, Stoner M, Safe S (2007) Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells. Toxicol Appl Pharm 223:28–38
Kjeldsen LS, Bonefeld-Jorgensen EC (2013) Perfluorinated compounds affect the function of sex hormone receptors. Environ Sci Pollut Res Int 20:8031–44
Kotake-Nara E, Saida K (2007) Characterization of CoCl2-induced reactive oxygen species (ROS): Inductions of neurite outgrowth and endothelin-2/vasoactive intestinal contractor in PC12 cells by CoCl2 are ROS dependent, but those by MnCl2 are not. Neurosci Lett 422:223–7
Krovel AV, Softeland L, Torstensen B, Olsvik PA (2008) Transcriptional effects of PFOS in isolated hepatocytes from Atlantic salmon (Salmo salar L.). Comput Biochem Phys C 148:14–22
Leaver MJ, Clarke DJ, George SG (1992) Molecular studies of the phase II xenobiotic-conjugating enzymes of Pleuronectid flatfish. Aquat Toxicol 22:265–278
Liu XH, Kirschenbaum A, Yao S, Stearns M, Holland J, Claffey K, Levine A (1999) Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line. Clin Exp Metastasis 17:687–694
Liu C, Du Y, Zhou B (2007) Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes. Aquat Toxicol 85:267–77
Liu Y, Ma Z, Zhao C, Wang Y, Wu G, Xiao J, McClain CJ, Li X, Feng W (2014) HIF-1α and HIF-2α are critically involved in hypoxia-induced lipid accumulation in hepatocytes through reducing PGC-1α-mediated fatty acid β-oxidation. Toxicol Lett 226:117–123
Livingstone DR (2001) Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar Pollut Bull 42:656–66
Lushchak VI, Bagnyukova TV (2006) Effects of different environmental oxygen levels on free radical processes in fish. Comp Biochem Physiol B 144:283–9
Lushchak VI, Bagnyukova TV (2007) Hypoxia induces oxidative stress in tissues of a goby, the rotan Perccottus glenii. Comp Biochem Physiol B 148:390–7
Lushchak VI, Bagnyukova TV, Lushchak OV, Storey JM, Storey KB (2005) Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues. Int J Biochem Cell Biol 37:1319–30
Mansfield KD, Guzy RD, Pan Y, Young RM, Cash TP, Schumacker PT, Simon MC (2005) Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-α activation. Cell Metab 1:393–399
Michaelidis B, Spring A, Pörtner HO (2007) Effects of long-term acclimation to environmental hypercapnia on extracellular acid–base status and metabolic capacity in mediterranean fish Sparus aurata. Mar Biol 150:1417–1429
Miller MJ (1989) Syntheses and therapeutic potential of hydroxamic acid based siderophores and analogs. Chem Rev 89:1563–1579
Monostory K, Jemnitz K, Vereczkey L (1996) Xenobiotic metabolizing enzymes in fish: diversity, regulation and biomarkers for pollutant exposure. Acta Physiol Hung 84:369–81
Mortensen AS, Arukwe A (2007) Interactions between estrogen- and ah-receptor signalling pathways in primary culture of salmon hepatocytes exposed to nonylphenol and 3,3′,4,4′-tetrachlorobiphenyl (congener 77). Comp Hepatol 6:2
Munday PL, McCormick MI, Nilsson GE (2012) Impact of global warming and rising CO2 levels on coral reef fishes: what hope for the future? J Exp Biol 215:3865–3873
Nathan C, Cunningham-Bussel A (2013) Beyond oxidative stress: an immunologist’s guide to reactive oxygen species. Nat Rev Immunol 13:349–361
Nebert DW, Gonzalez FJ (1987) P450 genes: structure, evolution, and regulation. Annu Rev Biochem 56:945–993
Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert D (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1–42
Obenour DR, Michalak AM, Zhou Y, Scavia D (2012) Quantifying the impacts of stratification and nutrient loading on hypoxia in the northern Gulf of Mexico. Environ Sci Technol 46:5489–96
Olsvik PA, Vikesa V, Lie KK, Hevroy EM (2013) Transcriptional responses to temperature and low oxygen stress in Atlantic salmon studied with next-generation sequencing technology. BMC Genomics 14:817
Olufsen M, Cangialosi MV, Arukwe A (2014) Modulation of membrane lipid composition and homeostasis in salmon hepatocytes exposed to hypoxia and perfluorooctane sulfonamide, given singly or in combination. Plos One 9:e102485
Osburn WO, Kensler TW (2008) Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults. Mutat Res 659:31–9
Pankhurst NW, Munday PL (2011) Effects of climate change on fish reproduction and early life history stages. Mar Freshw Res 62:1015–1026
Pesonen M, Andersson T (1987) Subcellular localization and properties of cytochrome P-450 and UDP glucuronosyltransferase in the rainbow trout kidney. Biochem Pharmacol 36:823–9
Prevedouros K, Cousins IT, Buck RC, Korzeniowski SH (2006) Sources, fate and transport of perfluorocarboxylates. Environ Sci Technol 40:32–44
Rahman MS, Thomas P (2012) Effects of hypoxia exposure on hepatic cytochrome P450 1A (CYP1A) expression in Atlantic croaker: molecular mechanisms of CYP1A down-regulation. Plos One 7:e40825
Regoli F, Giuliani ME (2014) Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Mar Environ Res 93:106–17
Regoli F, Pellegrini D, Cicero AM et al (2014) A multidisciplinary weight of evidence approach for environmental risk assessment at the Costa Concordia wreck: integrative indices from Mussel Watch. Mar Environ Res 96:92–104
Rimoldi S, Terova G, Ceccuzzi P, Marelli S, Antonini M, Saroglia M (2012) HIF-1α mRNA levels in Eurasian perch (Perca fluviatilis) exposed to acute and chronic hypoxia. Mol Biol Rep 39:4009–4015
Rissanen E, Tranberg HK, Sollid J, Nilsson GE, Nikinmaa M (2006) Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a poikilothermic vertebrate, crucian carp (Carassius carassius). J Exp Biol 209:994–1003
Scavia D, Evans MA, Obenour DR (2013) A scenario and forecast model for Gulf of Mexico hypoxic area and volume. Environ Sci Technol 47:10423–8
Schreck CB (2010) Stress and fish reproduction: the roles of allostasis and hormesis. Gen Comput Endocrinol 165:549–556
Schreck CB, Contreras-Sanchez W, Fitzpatrick MS (2001) Effects of stress on fish reproduction, gamete quality, and progeny. Aquaculture 197:3–24
Shang EH, Yu RM, Wu RS (2006) Hypoxia affects sex differentiation and development, leading to a male-dominated population in zebrafish (Danio rerio). Environ Sci Technol 40:3118–3122
Slotkin TA, MacKillop EA, Melnick RL, Thayer KA, Seidler FJ (2008) Developmental neurotoxicity of perfluorinated chemicals modeled in vitro. Environ Health Perspect 116:716–22
Soitamo AJ, Råbergh CM, Gassmann M, Sistonen L, Nikinmaa M (2001) Characterization of a hypoxia-inducible factor (HIF-1α) from Rainbow trout. Accumulation of protein occurs at normal venous oxygen tension. J Biol Chem 276:19699–19705
Storey KB (1996) Oxidative stress: animal adaptations in nature. Braz J Med Biol Res 29:1715–33
Swedenborg E, Pongratz I, Gustafsson JA (2010) Endocrine disruptors targeting ER-beta function. Int J Androl 33:288–97
Terova G, Rimoldi S, Corà S, Bernardini G, Gornati R, Saroglia M (2008) Acute and chronic hypoxia affects HIF-1α mRNA levels in sea bass (Dicentrarchus labrax). Aquaculture 279:150–159
Thomas P, Rahman MS (2012) Extensive reproductive disruption, ovarian masculinization and aromatase suppression in Atlantic croaker in the northern Gulf of Mexico hypoxic zone. Proc Biol Sci 279:28–38
Tiemann U (2008) In vivo and in vitro effects of the organochlorine pesticides DDT, TCPM, methoxychlor, and lindane on the female reproductive tract of mammals: a review. Reprod Toxicol 25:316–26
Tomy GT, Tittlemier SA, Palace VP, Budakowski WR, Braekevelt E, Brinkworth L, Friesen K (2003) Biotransformation of N-ethyl perfluorooctanesulfonamide by rainbow trout (Onchorhynchus mykiss) liver microsomes. Environ Sci Technol 38:758–762
Tripathi RK, Mohindra V, Singh A, Kumar R, Mishra RM, Jena JK (2013) Physiological responses to acute experimental hypoxia in the air-breathing Indian catfish, Clarias batrachus (Linnaeus, 1758). J Biosci 38:373–83
von Ehrenstein OS, Fenton SE, Kato K, Kuklenyik Z, Calafat AM, Hines EP (2009) Polyfluoroalkyl chemicals in the serum and milk of breastfeeding women. Reprod Toxicol 27:239–45
Vorrink SU, Severson PL, Kulak MV, Futscher BW, Domann FE (2014) Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines. Toxicol Appl Pharmacol 274:408–416
Wågbø AM, Cangialosi MV, Cicero N, Letcher RJ, Arukwe A (2012) Perfluorooctane sulfonamide-mediated modulation of hepatocellular lipid homeostasis and oxidative stress responses in Atlantic salmon hepatocytes. Chem Res Toxicol 25:1253–1264
Wallace KB (1989) Glutathione-dependent metabolism in fish and rodents. Environ Toxicol Chem 8:1049–1055
Wenger RH (2002) Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 16:1151–62
White SS, Fenton SE, Hines EP (2011) Endocrine disrupting properties of perfluorooctanoic acid. J Steroid Biochem 127:16–26
Williams DE, Lech JJ, Buhler DR (1998) Xenobiotics and xenoestrogens in fish: modulation of cytochrome P450 and carcinogenesis. Mutat Res 399:179–92
Wu RSS (2009) Effects of hypoxia on fish reproduction and development. Fish Physiol 27:79–141
Wu RSS, Zhou BS, Randall DJ, Woo NYS, Lam PKS (2003) Aquatic hypoxia is an endocrine disruptor and impairs fish reproduction. Environ Sci Technol 37:1137–1141
Xu L, Krenitsky DM, Seacat AM, Butenhoff JL, Anders MW (2004) Biotransformation of N-ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide by rat liver microsomes, cytosol, and slices and by expressed rat and human cytochromes P450. Chem Res Toxicol 17:767–75
Zhang N, Walker MK (2007) Crosstalk between the aryl hydrocarbon receptor and hypoxia on the constitutive expression of cytochrome P4501A1 mRNA. Cardiovasc Toxicol 7:282–290
Acknowledgments
This study was funded by the Norwegian Research Council project number 1964442/S40.
Author information
Authors and Affiliations
Corresponding author
Additional information
Responsible editor: Cinta Porte
Rights and permissions
About this article
Cite this article
Olufsen, M., Arukwe, A. Endocrine, biotransformation, and oxidative stress responses in salmon hepatocytes exposed to chemically induced hypoxia and perfluorooctane sulfonamide (PFOSA), given singly or in combination. Environ Sci Pollut Res 22, 17350–17366 (2015). https://doi.org/10.1007/s11356-014-3847-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11356-014-3847-y